Western Blot Analysis of Bacterial Proteins

Bacteria pellets were resuspended in phosphate-buffered saline (PBS) with 1× final sample buffer (FSB) and, after boiling at 100°C, loaded on 12.5% SDS-PAGE. A protein molecular weight marker (page ruler; Thermo Fisher) was included in each electrophoresis run. Proteins were transferred onto nitrocellulose membranes (Hybond-P; Millipore) and filter incubated with mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich), rabbit polyclonal anti-OmpA, or anti-VirF halon (68 (link)) antibodies. Secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich). Signals were produced with ECL Star (Euroclone) and detected with ChemiDoc gel imaging system (Bio-Rad Laboratories). The densitometric analysis was performed by ImageJ software.

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Trirocco R., Pasqua M., Tramonti A., Grossi M., Colonna B., Paiardini A, & Prosseda G. (2023). Fatty Acids Abolish Shigella Virulence by Inhibiting Its Master Regulator, VirF. Microbiology Spectrum, 11(3), e00778-23.

Publication 2023

page ruler Hybond-P ECL Star ChemiDoc gel imaging system

A protein Anti igg Antibodies Bacteria Buffer Densitometric Electrophoresis Goat Halon Horseradish peroxidase Membranes Molecular marker Monoclonal antibody Mouse Nitrocellulose Pellets Phosphate Proteins Rabbit Saline Sds page Virf

Corresponding Organization : Sapienza University of Rome

Other organizations : National Research Council, Institute of Molecular Biology and Pathology

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Variable analysis

independent variables

Bacterial pellets resuspension in phosphate-buffered saline (PBS) with 1× final sample buffer (FSB)

dependent variables

Protein expression levels detected by western blot using anti-FLAG M2, anti-OmpA, and anti-VirF antibodies
Protein molecular weight determined by SDS-PAGE

control variables

Protein molecular weight marker (page ruler; Thermo Fisher) included in each electrophoresis run
Boiling of samples at 100°C prior to SDS-PAGE

controls

Positive control: Protein molecular weight marker
Negative control: Not explicitly mentioned

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